The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terns as provided for by the terms of contract CA75979, awarded by the National Cancer Institute of the National Institutes of Health.
Throughout the application various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in the application in order to more fully describe the state of the art to which this invention pertains.
1. Field of the Invention
The present invention is related to the biomedical arts, in particular to biotechnology.
2. Discussion of the Related Art
Pituitary tumor-transforming gene (PTTG) is a recently described oncogene isolated from pituitary tumor growth hormone-secreting cells by differential display. (Pei, L., et al., Isolation and characterization of a pituitary tumor-transforming gene (PTTG), Mol. Endo. 11:433–441 [1997]). PTTG is believed to be a securin protein and has 44.6% amino acid identity with Xenopus securin. (Zou, H., et al., Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis, Science 285:418–422 [1999]; Mei, J., Huang, X., and Zhang, P., Securin is not required for cellular viability, but is required for normal growth of mouse embryonic fibroblasts, Current Biology 11:1197–1201 [2001]).
PTTGs have been identified in rat, mouse, and human cells. (e.g., PCT/US97/21463; Wang, Z., et al., Pituitary tumor transforming gene (PTTG) transforming and transactivation activity, J. Biol. Chem. 275:7459–7461 [2000]). The human PTTG family consists of at least four homologous genes, of which PTTG1 is located on chromosome 5q33. (Prezant, T. R., et al., An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family, J. Clin. Endocrinol. Metab. 84:1149–1152 [1999]).
PTTG has been shown to upregulate basic fibroblast growth factor secretion (Zhang, X. et al., Structure, expression, and function of human pituitary tumor-transforming gene (PTTG), Mol. Endocrinol. 13:156–66 [1999a]), and transactivate DNA transcription (Dominguez, A. et al. [1998]; Wang, Z. et al., Pituitary tumor transforming gene (PTTG) transactivating and transforming activity, J. Biol. Chem. 275:7459–61[2000]).
Human PTTG1 is expressed at low levels in most normal human tissues. (Chen, L. et al., Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization, Gene. 248:41–50 [2000]; Heaney, A. P. et al. [2000]). PTTG is abundant only in normal testis and thymus. (Wang, Z., et al., Characterization of the murine pituitary tumor transforming gene (PTTG) and its promoter, Endocrinology 141:763–771 [2000]). When expressed at normal levels, PTTG mediates promoter transcriptional activation. (Wang, Z., et al., Pituitary tumor transforming gene (PTTG) transforming and transactivation activity, J. Biol. Chem. 275:7459–7461 [2000]). By dysregulating chromatid separation, PTTG overexpression also leads to aneuploidy, i.e., cells having one or a few chromosomes above or below the normal chromosome number (Zou et al. [1999]; Yu, R., et al., Pituitary tumor transforming gene (PTTG) regulates placental JEG-3 cell division and survival: evidence from live cell imaging, Mol. Endo. 14:1137–1146 [2000]). At the end of metaphase, securin is degraded by an anaphase-promoting complex, releasing tonic inhibition of separin, which in turn mediates degradation of cohesins, the proteins that hold sister chromatids together. Overexpression of a nondegradable PTTG disrupts sister chromatid separation (Zou et al. [1999]) and overexpression of PTTG causes apoptosis and inhibits mitoses (Yu, R. et al. [2000]). The securin function of PTTG suggests that PTTG may also be expressed in normal proliferating cells. In adult humans, PTTG1 mRNA is most abundant in testis, an organ containing rapidly proliferating gametes. (Zhang, X. et al. [1999a]); Wang, Z. et al. [2000]).
In contrast, PTTG1 is highly expressed in human tumors and is responsive to estrogen induction. (Zhang, X., et al., Structure, expression, and function of human pituitary tumor-transforming gene (PTTG), Mol. Endo. 13:156–166 [1999a]; Heaney, A. P., et al., Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis, Nature Med. 5:1317–1321 [1999]). Indeed, PTTG is highly expressed in pituitary tumors and neoplasms from the hematopoietic system and colon, and PTTG is considered to be a proto-oncogene, because PTTG overexpression in NIH3T3 cells induces cell transformation and in vivo tumor formation. (Pei, L., et al., Isolation and characterization of a pituitary tumor-transforming gene (PTTG), Mol. Endo. 11:433–441 [1997]; Zhang, X. et al. [1999a]; Zhang, X. et al., Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas, J. Clin. Endocrinol. Metab. 84:761–67 [1999b]; Heaney, A. P. et al., Pituitary tumor transforming gene in colorectal tumors, Lancet 355:712–15 [2000]; Dominguez, A. et al., hPTTG, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG, Oncogene 17:2187–93 [1998]; Saez, C. et al., hPTTG is over-expressed in pituitary adenomas and other primary epithelial neoplasias, Oncogene 18:5473-6 [1999]). In addition, it was recently observed that PTTG1 overexpression in rat FRTL5 thyroid cells and in human thyroid cell cultures caused in vitro transformation and produces a dedifferentiated neoplastic phenotype. (Heaney, et al., Transforming events in thyroid tumorigenesis and their association with follicular lesions, J. Clin. Endocrinol. Metab. 86(10):5025–5032 [2001]).
The recent discovery of a human PTTG gene 2, which shares high sequence homology with human PTTG1, implying the existence of a PTTG gene family. (Prezant, T. R., et al., An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family, J. Clin. Endocrinol. Metab. 84:1149–1152 [1999]). Murine PTTG shares 66% nucleotide base sequence homology with human PTTG1 and also exhibits transforming ability. (Wang, Z. and Melmed, S., Characterization of the murine pituitary tumor transforming gone (PTTG) and its promoter, Endocrinology [In Press; 2000]). A proline-rich region was identified near the protein C-terminus that is critical for PTTG1's transforming activity. (Zhang, X., et al. [1999a]), as demonstrated by the inhibitory effect on in vitro transformation, in vivo tumorigenesis, and transactivation, when point mutations were introduced into the proline-rich region. Proline-rich domains may function as SH3 binding sites to mediate signal transduction of protein-tyrosine kinase. (Pawson, T., Protein modules and signaling networks, Nature 373:573–580 [1995]; Kuriyan, J., and Cowburn, D., Modular peptide recognition domains in eukaryotic signaling, Annu, Rev. Biophys. Biomol. Struct. 26:259–288 [1997]).
The transforming ability of PTTG has given rise to much interest into the study of its role in tumorigenesis in various tissues and organs, including the pituitary and thyroid. Moreover, the correlation between sex steroid expression and tumorigenesis also has garnered much interest. In general, various distinct molecular events (Farid, N. R., et al., Molecular basis of thyroid cancer, Endocr. Rev. 15:202–232 [1994]) occurring in thyroid neoplasia, including ras mutations, detected early in both benign and malignant tumors, activation of TSH receptor, and Gsα-subunit mutation have been reported in some follicular carcinomas. (Surez H. G., et al., gsp mutations in human thyroid tumors, Oncogene 6:677-679 [1991].
TSH is an important thyroid growth factor, and the cooperative regulation of thyrocyte growth by TSH and sex steriods has been supported by both animal studies and epidemiological analyses. (Clark, O. H., et al., Estrogen and thyroid-stimulating hormone (TSH) receptors in neoplastic and nonneoplastic human thyroid tissues, J. Surg. Res. 38:89-96 [1985]; Mori M, et al., Effects of sex difference, gonadectomy, and estrogen on N-methyl-N-nitrosourea induced rat thyroid tumors, Cancer Res 50(23):7662–7 [1990]).
Moreover, in addition to induction of PTTG expression in pituitary cells, estrogen administration also has been reported to increase the mean serum TSH levels in the pituitary and thyroid glands of female rats. (Mori M, et al., Effects of sex difference, gonadectomy, and estrogen on N-methyl-N-nitrosourea induced rat thyroid tumors, Cancer Res 50(23):7662-7 [1990]). This study also suggested a complex interplay between sex steroids and TSH on thyroid cell growth, where both male and female rats were more susceptible to radiation induced thyroid tumors in the presence of TSH stimulation, while ovariectomy or castration attenuated thyroid tumor occurrence. The complex interplay between TSH and PTTG also has been shown. That is, in a recent study, it was observed that TSH treatment of rat FRTL5 cells or primary human thyroid cells induced PTTG expression in vitro and administration of estrogen to rats induced rat thyroidal PTTG expression. (Heaney, et al., Transforming events in thyroid tumorigenesis and their association with follicular lesions, J. Clin. Endocrinol. Metab. 86(10):5025–5032 [2001]).
With the interest in the study of the etiology of various cancers follows the development of various treatment strategies. For example, iodine was shown to inhibit thyroid cell growth and its uptake is believed to be facilitated by sodium/iodide symporter (NIS) in thyroid follicular cells, or FRTL5 cells. Furthermore, the use of radioactive isotopes of iodine, such as 125I and 131I have been used to target tumor cells with high NIS gene expression.
It is also known that TSH can increase iodide uptake in a dose-dependent manner, and this effect is correlated with a rapid increase in NIS gene expression. Estradiol has been shown to block TSH-induced sodium/iodide symporter (NIS) expression, and treatment of cells with estradiol together with an estrogen receptor antagonist restored TSH-induced NIS expression to normal levels. (Furlanetto, T. W., et al., Estradiol Increases Proliferation and Down-Regulates the Sodium/Iodide Symporter Gene in FRTL-5 Cells1, Endocrinology 140(12): 5705–5711 [1999]; Furlanetto, T. W., et al., Estradiol decreases iodide uptake by rat thyroid follicular FRTL-5 cells, Braz J Med Biol Res 34(2) 259–263 [2001]). In contrast, a recent study showed that overexpression of wild-type PTTG1 in normal human thyroid cells in vitro and rat FRTL5 cells exhibited decreased 125I, uptake compared with controls. (Heaney, et al., Transforming events in thyroid tumorigenesis and their association with follicular lesions, J. Clin. Endocrinol. Metab. 86(10):5025–5032 [2001]).
To date, the development of an appropriate cell model for the study of the consequences of varying degrees of PTTG expression has been limited. Thus, there is a need in the art for a cellular model wherein PTTG expression, among other gene products, may be modulated and the resulting effects may be studied.